1. We are studying the structure, function, and expression of the transmembrane channel-like (TMC) genes 1 and 2. We have successfully generated mice segregating knockout (null) alleles of Tmc1 and Tmc2. We are characterizing their mutant auditory and vestibular (balance) phenotypes at the functional, cellular and molecular levels. We are studying genetic interactions between Tmc1 and Tmc2 in mice carrrying knockout alleles of both genes. We are generating new anti-TMC1 and anti-TMC2 antibodies by in vivo and vivo methods and will use the corresponding knockout mice to test the validity of staining patterns of antibodies to Tmc1 and Tmc2 in inner ear sensory hair cells. [unreadable] 2. We completed a study defining the transmembrane topologic organization of TMC1 protein expressed in tissue culture cells. Our results indicate that it is a six-pass transmembrane protein with cytoplasmically oriented N- and C-termini. [unreadable] 3. We previously used a yeast two-hybrid screen to isolate genes encoding proteins that potentially interact with Tmc1 and Tmc2. We narrowed the list to a few candidate genes implicated in vesicular trafficking and in antiapoptosis and cell survival. We are are continuing to use a combination of approaches to determine which interactions might occur in situ in hair cells.[unreadable] 4. We have completed a mutation analysis of TMC1 in 10 Pakistani families segregating recessive deafness linked to markers near TMC1. We identified several new amino acid substitution mutations that will be useful as negative controls for studies of TMC1 protein function. We extended the results of our previous study showing that 5% of recessive deafness in Pakistan is caused by TMC1 mutations. On half of those cases are caused by the same nonsense mutation, R34X, that was derived from a common ancestor. This will facilitate diagnostic strategies for deafness in individuals from Pakistan and south Asia. We have also screened large numbers of DNA samples from a Deafness DNA Repository in order to define the contribution of some of these common south Asian mutations to deafness in individuals from other ethnic groups.[unreadable] 5. In collaboration with the LMG, we showed that the Usher/deafness gene product protocadherin 15 is the tip link antigen of hair cell stereocilia. This is an important discovery since the tip link is thought to be the structure that mechanically gates the mechanotransduction channel of inner ear hair cells.[unreadable] 6. We have generated knockout mice for Tmc3, Tmc6, and Tmc8 to better understand the function(s) of Tmc genes and proteins. Tmc3 appears to be expressed primarily in neuroendocrine tissues and organs. Tmc6 and Tmc8 are primarily expressed in lymphoid cells and tissues, and truncating allele of either TMC6 or TMC8 cause the autosomal recessive disease epidermodysplasia verruciformis, characterized by chronic cutaneous HPV infections and susceptibility to non-melanoma skin cancers, and defects in helper T cell function. We have now generated mice transmitting knockout alleles of Tmc3, Tmc6, Tmc8 and a Tmc6/Tmc8 double knockout (the genes lie next to each other on chr. 17) in their germline and are currently expanding the colonies to generate mice for experimental analysis. [unreadable] 7. We are continuing our effort to identify the gene mutated in the mouse Twirler strain. Heterozygous Twirler mice have inner ear malformations and obesity, whereas homozygous mice are born with cleft palate and die at birth. The critical interval containing the Twirler gene is approximately 750 kilobases and contains one known gene and two predicted genes. We have completed nucleotide sequence analysis all of the exons and flanking intronic sequences in these genes and not found a pathogenic mutation. We have identified different splice isoforrm variants encoded by the Twirler allele of one of the predicted genes and we are carefully searching for mutations in the non-coding region of that gene. [unreadable] 8. Enlargement of the vestibular aqueduct (EVA) is the most commonly detected radiologic malformation in temporal bones of individuals with hearing loss. A significant proportion of EVA cases have been reported to be associated with mutations of the SLC26A4 gene, in which mutations cause Pendred syndrome. PS is an autosomal recessive disorder comprised of bilateral sensorineural hearing loss and a defect in the ability of the thyroid gland to organify iodine. EVA is a universal finding in the ears of affected PS individuals. PS is correlated with two mutant SLC26A4 alleles, and nonsyndromic EVA is associated with one or zero mutant SLC26A4 alleles. Based upon our data, we hypothesize that one or more other genetic or environmental factors may act alone or in combination with a single SLC26A4 mutation to cause EVA. We have analyzed a number of candidate genes based upon known association with EVA in other hearing loss syndromes, or based upon molecular function, as candidates for the etiologic cofactor. We have not identified any convincing genetic cofactors by this approach, and are now actively accruing families with two or more individuals with EVA to perform an allele-sharing genomewide screen for such cofactors. We are analyzing our genotype and phenotype data to identify clinical features that may guide molecular diagnosis or clinical prognosis. We have completed functional studies of several missense substitutions that have been always or usually been detected as the single SLC26A4 variant in EVA patients and shown that they appear to have wild type trafficking and functional activity, indicating they are benign polymorphic variants. These results have important implications for molecular diagnosis of EVA patients, as well as for categorizing patients according to SLC26A4 genotype for studies to identify other causes of EVA.[unreadable] 9. We completed a study of a large Caucasian family segregating autosomal dominant, nonsyndromic, postlingual-onset, progressive sensorineural hearing loss. We determined that their hearing loss was linked to markers at the DFNA10 locus. Nonsyndromic hearing loss DFNA10, as well as a syndromic form of hearing loss that includes dilated cardiomyopathy, are caused by truncating mutations of the EYA4 gene. We sequenced EYA4 and found that this family has a frameshift mutation. We carefully studied the cardiac phenotypes of affected individuals and showed that they do not have any evidence of cardiomyopathy. Our results confirm a previously proposed correlation of the position of truncating EYA4 mutations with the presence or absence of cardiomyopathy. Our study also showed there does not seem to be a similar correlation of mutation position with the onset or severity of hearing loss.[unreadable] 10. We completed a temporal bone histopathologic and genetic study of an infant with keratitis-ichthyosis-deafness syndrome. We showed that this child was deaf due to developmental dysplasia of the cochlear epithelium associated with a mutation of GJB2, which encodes connexin 26. Our result indicates that at least some GJB2 mutations cause deafness by disrupting cochlear development.[unreadable] 11. We ascertained a large North American family segregating progressive, nonsyndromic sensorineural hearing loss in a matrilineal/maternal/mitochondrial pattern of inheritance. We sequenced the entire mitochondrial genome in several affected individuals and have identified a rare mutation in the tRNA-Ser(UCN) gene.